Cigarette smoking is a major risk factor of human cancers. Human cytochrome P4502A6 (CYP2A6) plays a predominant role in the metabolic activation of N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK), two major carcinogenic tobacco-specific nitrosamines, as well as in the metabolism of nicotine which is responsible for smoking addiction. Existence of functional genetic polymorphism of CYP2A6 is strongly suggested by the reports on large inter-individual variations (up to 170-fold) in CYP2A6 activity (assayed as coumarin 7-hydroxylase) in general populations, and on bimodal activity distribution. There area a total of 6 genetic variants on human CYP2A6, including 3 missense variants we recently identified. One of the reported missence variants (2A6v1, leu160His) lacked coumarin 7-hydroxylase activity. However, the activities of 2A6v1 and other variants in metabolizing NNN, NNK, and nicotine as well as their importance in tobacco-related carcinogenesis have not been investigated. Our working hypothesis is that functional genetic polymorphisms of CYP2A6, such as 2A6v1, significantly affects an individual's ability to metabolize NNN, NNK, and nicotine. Therefore, it could be an important determinant in human susceptibility to tobacco-related carcinogenesis. As an essential step to our working hypothesis, the present proposal focuses on the functional characterization of CYP2A6 genetic variants with the following specific aims: 1. To characterize the functional significance of CYP2A6 missense genetic variants through: (a) generation of the variant proteins by site-directed mutagenesis and heterologous expression; (b) comparison of the enzyme kinetics of the variants with the wild-type protein in metabolizing NNN, NNK and nicotine; (c) comparison of NNN- or NNK-induced cytotoxicity in cells transfected with either wild-type or variant CYP2A6 cDNAs; and (d) determination of protein stability of the missense variants. 2. To determine the allelic frequency distribution of functional CYP2A6 polymorphisms in different ethnic populations. This study will provide background information for future epidemiological studies, and provide the genotyped subjects for the human study proposed in specific aim 3. 3. To determine the impact of functional YCP2A6 genetic polymorphisms on the in vivo metabolism of nicotine and coumarin, both probe drugs of CYP2A6, by correlating the urinary metabolic ratio (MR) of nicotine and coumarin with the CYP2A6 polymorphic genotypes in volunteer subjects. Individuals with significant MR alterations not without known CYP2A6 polymorphisms will be used to identify new CYP2A6 variants. Our proposed studies are critical to elucidate the functional importance of CYP2A6 genetic polymorphism and help asses the possibility of using CYP2A6 genetic polymorphism as a susceptibility marker of tobacco-related human carcinogenesis, which could contribute significantly to cancer prevention by identifying susceptible subpopulations.